Advancements in stem cell research now suggest potential applications ranging from bone [1-3] and cartilage [4] repair, to the treatment of heart attacks [5-7] and strokes [8,9]. With this ever-broadening array of promising therapies, the use of animal products in stem cell culture emerges as a common hurdle. Significant cell expansion in vitro has typically relied on media supplemented with largely uncharacterized animal sera. The clinical use of cells exposed to such sera presents numerous safety issues. Xenogeneic antigens introduced in this way are internalized by cells in culture and cannot be eliminated despite multiple washes in buffered saline solutions [10]. These foreign antigens have been known to trigger significant immune reactions clinically. Human cells cultured in fetal calf serum (“FCS”), and subsequently transplanted into patients have induced severe anaphylaxis [11], and arthus-like immune reactions consistent with type III hypersensitivity reactions [12]. Additional reports of the sustained elevation of anti-FCS antibodies indicate a maintained sensitively to cells exposed to FCS. This unintended “vaccination” against the transplanted cells expressing FCS antigens may be partly responsible for the decreased durability observed with in gene therapy trials, particularly when multiple doses of FCS cultured cells are given [13]. The elimination of this undefined and potentially dangerous component in cell culture media would reduce the exposure of patients to xenogeneic pathogens and allow for multiple cell administrations without the risk of a stimulated immune response or in the worst case, anaphylaxis.
Mesenchymal stem cells in general, and adipose stem cells in particular, hold great promise for future clinical therapies which enhance the body's natural ability to heal itself. One hurdle common to the use of these potential therapies is the current practice of using fetal bovine serum or other animal sera in the culture media of cells intended for use in humans. The undefined and variable nature of animal sera, as well as the associated risk of introducing xenobiotic pathogens and triggering severe allergic responses in some subjects, presents a technical problem presently unresolved in the field.
In recent years, the identification of mesenchymal stem cells, chiefly obtained from bone marrow, has led to advances in tissue regrowth and differentiation. Such cells are pluripotent cells found in bone marrow and periosteum, and they are capable of differentiating into various mesenchymal or connective tissues. For example, such bone-marrow derived stem cells can be induced to develop into myocytes upon exposure to agents such as 5-azacytidine (Wakitani et al., Muscle Nerve, 18 (12), 1417-26 (1995)). It has been suggested that such cells are useful for repair of tissues such as cartilage, fat, and bone (see, e.g., U.S. Pat. Nos. 5,908,784, 5,906,934, 5,827,740, 5,827,735), and that they also have applications through genetic modification (see, e.g., U.S. Pat. No. 5,591,625). While the identification of such cells has led to advances in tissue regrowth and differentiation, the use of such cells is hampered by several technical hurdles. One drawback to the use of such cells is that they are very rare (representing as few as 1/2,000,000 cells), making any process for obtaining and isolating them difficult and costly. Of course, bone marrow harvest is universally painful to the donor. Moreover, such cells are difficult to culture without inducing differentiation, unless specifically screened sera lots are used, adding further cost and labor to the use of such stem cells. U.S. Pat. No. 6,200,606 by Peterson et al., describes the isolation of CD34+ bone or cartilage precursor cells (of mesodermal origin) from tissues, including adipose.
The presence of adult multipotent “stem” cells has been demonstrated in a large number of tissues, for example the bone marrow, blood, liver, muscle, the nervous system, and in adipose tissue. Adult “stem” cells, which in theory are capable of infinite self-renewal, have great cell plasticity, i.e. the ability to differentiate into tissues other than those for which it was believed they were destined. The properties of said cells, which are similar to those of embryonic stem cells (ES), open up considerable therapeutic perspectives especially as their use does not pose the problems of compatibility and ethics, encountered with ES cells.
Adipose tissue plays an important and overlooked role in the normal development and physiology of humans and other mammalian species. Many different kinds of fat exist. The most common type is white adipose tissue, located under the skin (subcutaneous fat), within the abdominal cavity (visceral fat) and around the reproductive organs (gonadal fat). Less common in the adult human is brown adipose tissue, which plays an important role in generating heat during the neonatal period; this type of fat is located between the shoulder blades (interscapular), around the major vessels and heart (periaortic and pericardial), and above the kidney (suprarenal).
As women mature, they develop increased amounts of mammary adipose tissue. The mammary fat pad serves as an energy source during periods of lactation. Indeed, reproductive capacity and maturation are closely linked to the adipose tissue stores of the individual. Puberty in women and men correlates closely with the production and release of leptin, an adipose tissue derived hormone, and to body fat composition. Other adipose tissue sites play a structural role in the body. For example, the mechanical fat pads in the soles of the feet provide a cushion against the impact of walking. Loss of this fat depot leads to progressive musculoskeletal damage and impaired mobility. Bone marrow fat cells are present in bone marrow to provide energy to developing blood cells within the marrow. Bone marrow adipocytes are different than adipocytes present in adipose tissue, differing in morphology, physiology, biochemistry as well as their response to various stimulators such as insulin. Adipocytes present in bone marrow stroma may function to: 1) regulate the volume of hemodynamically active marrow; 2) serve as a reservoir for lipids needed in marrow cell proliferation, and 3) may be developmentally related to other cell lineages such as osteoblasts. White adipose tissue (i.e. body fat) in contrast, is involved in lipid metabolism and energy homeostasis (Gimble, The New Biologist 2(4), 1990, pp. 304-312).
There is a long felt need in the art for methods to identify, select, grow, and induce differentiation of adipose tissue-derived stem cells. The present invention satisfies these needs.